BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8’s affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.

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I look forward to seeing a revised form of your manuscript when it is ready and please let me know if you have questions or comments regarding the revision.In this study the authors investigate the chaperone requirements for connecting piece assembly and the relationship between BAG5, HSPAs and SPATA6.The authors establish a fertility defect for BAG5 mice and reveal mechanistic insight for molecular function that will also be valuable for understanding the role of BAG5 in other tissues, such as the heart (cardiac myopathy).The paper builds on previous work showing that disrupting SPATA6, a protein required for connecting piece assembly, can lead to acephalic spermatozoa.
The immunoprecipitation, mass spectrometry and validation approach shown in Fig 1 is well explained, and the fertility data of the BAG5 knockout mice are compelling.
I am not convinced by the data investigating colocalization of BAG5 with ER chaperones (in Fig 1 H-J and pg 7).The staining data is not sufficiently high resolution to discriminate between the ER and cytosol (and other organelles).Whilst it is possible that BAG5 is recruited to the cytosolic face of the ER, further evidence is required to demonstrate genuine import into the ER lumen in the absence of a targeting signal.HEK293 cells will also lack some of the specialist machinery of spermatids.The text on pg 7 should be revised to moderate the conclusions.
Similarly, I am not convinced by the HSPA8 co-localisation data and explanation (Fig 7E and pg 15).According to multiple papers, HSPA8 is cytosolically localised.The interpretation of the data therefore requires moderating.One HSPA8 role is in chaperone mediated autophagy to promote import of substrates into lysosomes.Have the authors considered that HSPA8 may be participating in the development of the acrosome, where some ER chaperones can accumulate?Pg 11: The authors state that "Together, these data hint that BAG5 interacts with SPATA6 to regulate the attachment of Cp and Sc to HTCA during spermiogenesis."However, there are other possibilities and primary data is lacking in full support of this assertion.For example, BAG5 could be working indirectly on unidentified clients in spermatids via the Hsp70 cycle, or could be influencing proteostasis through its role as a ubiquitin system modulator.
On page 13, why were HeLa cells chosen for the BAG5 cell line knockout?A myocyte (muscle) cell line might have been better to identify more physiologically/disease relevant protein interaction network.
The interaction experiments in the presence of ATP (pg 17 and Fig 8) support the notion that the NBD domain of HSPA8 is required for the phenotype, but a more thorough experiment would be to include non-hydrolysable analogs and to employ a nucleotide binding domain mutant.
Minor point: there are some spelling mistakes and grammar issues.The paper would benefit from additional editing for English language.

Review comments
The authors discovered that BAG5 complexes are essential for the head-tail coupling apparatus.They found that BAG5 regulates the chaperone protein HSPA8 and its loss causes abnormal protein misfolding.The present paper is a very excellent in the view of revealing BAG5 is essential for male fertility and also the molecular mechanisms underlying the development of the head-tail coupling apparatus.In addition, there is no major problem with the main experimental results or story, so I think it should be published in an EMBO report.However, the authors may be mistaken about cytoplasmic behavior in spermiogenesis.Due to this, I cannot ignore the schematic diagram in Fig 4E, movie EV3 and 4, as they might spread false information.In addition, discussion is very long and boring as it was just a list of contents from the past literature.Discussion should be systematically focused on information directly related to this literature.An excellent set of data is ruined by a boring discussion.

Major concerns
The diagram in Fig 4E, movies EV3 and EV4 must be modified.I have no complaints about head, acrosome, HTCA, and flagellar behavior.But the behavior of cytoplasm is totally strange.To my knowledge, I only know the following literature that systematically describes cytoplasmic behavior in spermatids.The authors should read page 20-27 in the book.Diagrams and movies lack views of cytoplasmic invagination, nuclear movement toward the cell surface, cytoplasmic lobes, and residual bodies.This paper could be read by many people, but I cannot ignore the possibility of spreading incorrect views, even schematic models.Russell, L., Ettlin, R., Sinha Hikim, A., and Clegg, E. (1990).Histological and histopathological evaluation of the testis.1990 (Cache River Press).
As mentioned in the preamble, the discussion is too long with a lot of listing of past literature contents.Please put yourself in the reader's shoes and make the contents more informative.

Minor concerns
It is difficult to see blue on a black background.I think the blue color should be changed to another color.Figs.1C, 1J, 4A and 7F are particularly difficult to see.
The pictures in Fig. 1E are too small.In contrast, Fig. 1F is too big and not so important.In addition, Fig. 1E contains data from KO mice even though they will be generated later.To demonstrate the significant data clearly, Fig 1E should be enlarged to show the localization of BAG5 in basal plate.This should be performed even if KO and 1F pictures are removed.In addition, steps 9 and 14 spermatids are too different in form.Therefore, the steps for spermatids should be the same.Endpieces in Fig. 3H are cross-sections of principal piece because they have fibrous sheath.In my experience, it is very difficult to find an endpiece with a clear axonemal structure in TEM.The endpiece of this figure should be removed.
All KO spermatids are oriented abnormally in Fig 4A (right-most) schematic.However, this figure may be misleading because less than 15% of all sperm are oriented abnormally.In addition, the author has not clarified experimentally the orientation of the sperm flagellum.Since the sperm flagellum orientation may be normal, the flagellum orientation should also be examined if this schematic is included.
Although this is a matter of personal interest, I suspect that cytoplasmic invagination does not occur in KO spermatids when looking at TEM images in Fig. 4C.As observed in WT spermatids, the tail end of centrosome should be close to cell surface.This is caused by cytoplasmic invagination.Since no cytoplasmic surface is seen at the posterior end of HTCA in KO spermatozoa, there must be an abnormality in cytoplasmic invagination.
What is "M" in Fig. 8H?There is no explanation (maybe manchette).ER can be observed outside the manchette in Fig. 8H.Do you have any evidence for it?

POINT-BY-POINT RESPONSE TO REVIEWERS
Our specific comments to each reviewer follow.Please note: Reviewer comments are in italics.Our responses are in blue font.The comments from the Reviewers have not been edited.We thank all Reviewers' comments, which enabled us to significantly improve the manuscript and highlight the importance of our study.

Referee #1:
In this study the authors investigate the chaperone requirements for connecting piece assembly and the relationship between BAG5, HSPAs and SPATA6.The authors establish a fertility defect for BAG5 mice and reveal mechanistic insight for molecular function that will also be valuable for understanding the role of BAG5 in other tissues, such as the heart (cardiac myopathy).The paper builds on previous work showing that disrupting SPATA6, a protein required for connecting piece assembly, can lead to acephalic spermatozoa.The immunoprecipitation, mass spectrometry and validation approach shown in Fig 1 is well explained, and the fertility data of the BAG5 knockout mice are compelling.
Thank you very much for your evaluation and valuable comments, which enabled us to improve the quality of the manuscript.

I am not convinced by the data investigating colocalization of BAG5 with ER chaperones (in Fig 1 H-J and pg 7)
. The staining data is not sufficiently high resolution to discriminate between the ER and cytosol (and other organelles).Whilst it is possible that BAG5 is recruited to the cytosolic face of the ER, further evidence is required to demonstrate genuine import into the ER lumen in the absence of a targeting signal.HEK293 cells will also lack some of the specialist machinery of spermatids.The text on pg 7 should be revised to moderate the conclusions.
Thank you for these helpful comments.To provide more conclusive evidence that BAG5 is located in the ER, we extracted the ER proteins from the mouse testes using the commercial Kit.The new results showed that BAG5 was indeed localized in the ER.We have shown these results as a new Figure 1I and revised the text accordingly (See lines 162-164).We have also revised the conclusions based on the additional experimental results (See lines 164-167 with yellow color highlighted).
Similarly, I am not convinced by the HSPA8 co-localisation data and explanation (Fig 7E and pg 15).According to multiple papers, HSPA8 is cytosolically localised.The interpretation of the data therefore requires moderating.
Thanks for pointing out this issue.We agreed with the notion that HSPA8 is cytosolically localized.We have repeated the immunostaining experiments for HSPA8 and BAG5 27th Nov 2023 1st Authors' Response to Reviewers localization in HEK293 cells and testicular sections and found that HSPA8 was indeed colocalized with BAG5 in the cytoplasm of HEK293 cells and the manchette of elongating spermatids, respectively (See new Figure 7E and lines 363-365 with yellow color highlighted).
One HSPA8 role is in chaperone mediated autophagy to promote import of substrates into lysosomes.Have the authors considered that HSPA8 may be participating in the development of the acrosome, where some ER chaperones can accumulate?
Thank you for your constructive thoughts.As we observed in Figure EV2E-F of this study, the acrosome developmental process was normal after the knockout of BAG5, which indicates that HSPA8-mediated protein folding activated by BAG5 was not associated with acrosome development.
Pg 11: The authors state that "Together, these data hint that BAG5 interacts with SPATA6 to regulate the attachment of Cp and Sc to HTCA during spermiogenesis."However, there are other possibilities and primary data is lacking in full support of this assertion.For example, BAG5 could be working indirectly on unidentified clients in spermatids via the Hsp70 cycle, or could be influencing proteostasis through its role as a ubiquitin system modulator.
Thanks for pointing out this issue.We agree with the other possible roles of BAG5 in spermiogenesis raised by this Reviewer, and it provided us with a future direction to investigate the underlying molecular regulation of BAG5 on spermatid development in a separate study.To clarify the statement in the current study, we have revised the text description in lines 279-280 accordingly.

On page 13, why were HeLa cells chosen for the BAG5 cell line knockout? A myocyte (muscle) cell line might have been better to identify more physiologically/disease relevant protein interaction network.
Thank you for raising this question.We also agree that the myocyte (muscle) cell line may be better for studying physiologically/disease-relevant protein interaction networks.However, the current study mainly focuses on the function of BAG5 in the reproductive system; therefore, we chose the reproductive system cell lines (such as HeLa) to verify the protein interaction in vitro.
The interaction experiments in the presence of ATP (pg 17 and Fig 8) support the notion that the NBD domain of HSPA8 is required for the phenotype, but a more thorough experiment would be to include non-hydrolysable analogs and to employ a nucleotide binding domain mutant.
Thank you for your careful review and great suggestions.As suggested, we performed the additional experiments and showed the new data in Figure 8F-I.The new results support the point again that the NBD domain of HSPA8 is required for the phenotype.We have also added the elaboration into text in lines 394-399 with yellow color highlighted.
Minor point: there are some spelling mistakes and grammar issues.The paper would benefit from additional editing for English language.
The manuscript has been checked and polished by a professional editing service.All typos and grammar issues have been corrected as far as possible.Thank you so much for this careful review.

Referee #2: Review comments
The authors discovered that BAG5 complexes are essential for the head-tail coupling apparatus.They found that BAG5 regulates the chaperone protein HSPA8 and its loss causes abnormal protein misfolding.The present paper is a very excellent in the view of revealing BAG5 is essential for male fertility and also the molecular mechanisms underlying the development of the head-tail coupling apparatus.In addition, there is no major problem with the main experimental results or story, so I think it should be published in an EMBO report.
Thank you very much for your appreciation of our work.

However, the authors may be mistaken about cytoplasmic behavior in spermiogenesis. Due to this, I cannot ignore the schematic diagram in Fig 4E, movie EV3 and 4, as they might spread false information.
We apologize for this mistake and appreciate that you have raised this critical comment.We have redrawn the schematic to show the cytoplasmic invagination and nuclear movement towards the cell surface in steps 6-8, and the formation and removal of cytoplasmic lobes to produce residual bodies in WT steps 15-16 of the new Figure 4E.Since it is difficult to visualize the developmental complex process of spermatids in spermiogenesis by 3D movie, we decided to remove the movies EV3-4 in the revision to avoid any misunderstanding for the readers.Thanks for your careful review.
In addition, discussion is very long and boring as it was just a list of contents from the past literature.Discussion should be systematically focused on information directly related to this literature.An excellent set of data is ruined by a boring discussion.
We agree with this suggestion and have rewritten the discussion section in the revision (See lines 427-470, 494-506, and 514-525 with yellow color highlighted).Thank you!

Major concerns The diagram in Fig 4E, movies EV3 and EV4 must be modified. I have no complaints about head, acrosome, HTCA, and flagellar behavior. But the behavior of cytoplasm is totally strange. To my knowledge, I only know the following literature that systematically describes cytoplasmic behavior in spermatids. The authors should read page 20-27 in the book.
Diagrams and movies lack views of cytoplasmic invagination, nuclear movement toward the cell surface, cytoplasmic lobes, and residual bodies.This paper could be read by many people, but I cannot ignore the possibility of spreading incorrect views, even schematic models.Russell, L., Ettlin, R., Sinha Hikim, A., and Clegg, E. (1990).Histological and histopathological evaluation of the testis. 1990 (Cache River Press).
Thank you for your comments and helpful guidance.We have read the recommended book carefully and gained a lot of knowledge regarding the process of spermiogenesis, especially on cytoplasmic invagination.We have corrected the schematic models in the new Figure 4E, including the cytoplasmic invagination, nuclear movement towards the cell surface, cytoplasmic lobes, and residual bodies.
As mentioned in the preamble, the discussion is too long with a lot of listing of past literature contents.Please put yourself in the reader's shoes and make the contents more informative.
Thank you for your suggestion.We have simplified the discussion to enhance its readability (See lines 427-470, 494-506, and 514-525).

Minor concerns
It is difficult to see blue on a black background.I think the blue color should be changed to another color.Figs.1C, 1J, 4A and 7F are particularly difficult to see.
Thank you for that excellent suggestion.As suggested, we changed the blue color to white color in Figures 1C, 1J (new 1H), 4A, and 7F (new 7E) and made the nucleus visible.
The pictures in Fig. 1E are too small.In contrast, Fig. 1F is too big and not so important.In addition, Fig. 1E contains data from KO mice even though they will be generated later.To demonstrate the significant data clearly, Fig 1E should be enlarged to show the localization of BAG5 in basal plate.This should be performed even if KO and 1F pictures are removed.In addition, steps 9 and 14 spermatids are too different in form.Therefore, the steps for spermatids should be the same.This is a good suggestion.We have enlarged Figure 1E to show the clear location of BAG5 in the Manchette and Basal plate, and we moved the KO data and Figure 1F to Appendix Figures S1 and S2.
Endpieces in Fig. 3H are cross-sections of principal piece because they have fibrous sheath.In my experience, it is very difficult to find an endpiece with a clear axonemal structure in TEM.The endpiece of this figure should be removed.
Thank you for your professional advice.We took the image of the endpiece carefully again and showed it in Figure 3H.
All KO spermatids are oriented abnormally in Fig 4A (right-most) schematic.However, this figure may be misleading because less than 15% of all sperm are oriented abnormally.In addition, the author has not clarified experimentally the orientation of the sperm flagellum.Since the sperm flagellum orientation may be normal, the flagellum orientation should also be examined if this schematic is included.
Thank you for your thoughtful comments.We have re-examined the orientation of the flagellum by co-immunostaining of AKAP4 (labels the fibrous sheath located in the principal piece) with ATP5A (marks the mitochondria located in the midpiece) and have shown the data in Appendix Figure S3.The new results showed that the midpiece (ATP5A, red) towards the basal membrane and the principal piece (AKAP4, green) close to the lumen, indicating that the orientation of the sperm flagellum was normal, as this Reviewer thought.We have, therefore, corrected the schematic diagram in Figure 4A (right-most) and revised the text accordingly (See lines 243-244 with yellow color highlighted).
Although this is a matter of personal interest, I suspect that cytoplasmic invagination does not occur in KO spermatids when looking at TEM images in Fig. 4C.As observed in WT spermatids, the tail end of centrosome should be close to cell surface.This is caused by cytoplasmic invagination.Since no cytoplasmic surface is seen at the posterior end of HTCA in KO spermatozoa, there must be an abnormality in cytoplasmic invagination.
Thank you for raising this interesting question.We agree with you and have drawn the schematic model to show the abnormal cytoplasmic invagination in KO spermatids in Figure 4E.We have also added this assumption in the revised text (See lines 256-261 with yellow color highlighted).
What is "M" in Fig. 8H?There is no explanation (maybe manchette).
Thank you for your careful review."M" is "manchette".We have added the explanation in the legend of Figure 8H.

ER can be observed outside the manchette in Fig. 8H. Do you have any evidence for it?
Thank you for raising this critical question.To ensure we provided the correct information for the readers, we reviewed the literature on the formation of germ cell organelles during spermiogenesis.Based on the published literature, ER undergoes marked changes and structural modifications during spermiogenesis (Clermont et al, 1993;Hermo et al, 2010;Toshimori et al, 2001).At the acrosome phase, the ER aligns along the inside and outside of manchette microtubules in the form of a fenestrated sleeve (Clermont et al., 1993;Clermont & Rambourg, 1978;Hermo et al., 2010;Kaneko et al, 2019;Nistal et al, 1980).Thus, we drew the schematic diagram in Figure 8H to show the abundant location of the ER close to the manchette.Thank you for the submission of your revised manuscript to EMBO reports.We have now received the full set of referee reports that is copied below.
As you will see, both referees conclude that the revision has strengthened the manuscript.However, referee 2 has some remaining concerns that I kindly ask you to address.I am also happy to discuss these experiments further with you.
From the editorial side, there are also a few things that we need before we can proceed with the official acceptance of your study.
-Please update the 'Conflict of interest' paragraph to our new 'Disclosure and competing interests statement'.For more information see https://www.embopress.org/page/journal/14693178/authorguide#conflictsofinterest-The author Xu Fan is listed as Xu Fan in the manuscript file but as Xv Fan in the submission system.Please correct either or.
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-The callout for Movie EV2 should be corrected: it should be "Movie EV1 and EV2" instead of "Movie EV1 and 2".
-The movie legends need to be removed from the manuscript file and provided as a readme.txtfile and then each should be zipped together with its corresponding video.
-Table EV1-EV5 are complex tables, please rename them to Dataset EV1-EV5 and correct the callouts in the manuscript.Please add a legend to the tab "Proteins" in Dataset EV1.Table EV6 would then be Table EV1 and Table EV7 becomes Table EV2.
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-The following sentence should be removed from the manuscript: "Expanded View Figures and Appendix File -The title should be removed from p45, (before the EV figure legends).
-We perform a routine figure check on all manuscripts before publication.In this context we noticed the following issues: A) Figure 2J -The image shown for the KO seems to be the same image as the WT, however rotated.This also applies to the source data images.Please check these images.Also, the cropped image does not fit with the bounding box for the WT.
B) It seems that some sections shown in Figure 4A have been used again in Figure EV2B (WT IX, KO IV).If this is the case, please clearly state this in the figure legend.C) Figure EV2 B KO an WT iii images look very similar to each other.Please check these.
-Source data need to be uploaded one folder per figure for main figures and can be grouped together for EV and Appendix.
-Our production/data editors have asked you to clarify several points in the figure legends (see below).Please incorporate these changes in the manuscript and return the revised file with tracked changes with your final manuscript submission.a) Please indicate the statistical test used for data analysis in the legends of figures 5a-b; 6a-b; 7a-b.b) Please note that information related to n is missing in the legend of figure 5a.c) Please note that the arrows are not defined in the legend of figure EV 5a.This needs to be rectified.
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We look forward to seeing a final version of your manuscript as soon as possible.Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive.We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored.Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly.BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice.In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion.Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins.Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.The authors have responded carefully to the points raised in review and have provided substantial new data, including a more detailed analysis of HSPA8 and the role of the NBD; and a more convincing localisation analysis of BAG5 and HSPA8.I have no further comments for the authors to address.

Referee #2:
Major points #1 The authors mentioned that KO spermatids have abnormal cytoplasmic invagination (L265).When I observed Fig. 4C, it looks abnormal.But cytoplasmic invagination could be found in KO spermatids (Fig. S1).I think this means there are no abnormalities in this process.You can check this process in the following paper.I recommend that you re-evaluate this process."CCDC183 is essential for cytoplasmic invagination around the flagellum during spermiogenesis and male fertility" by Shimada et al.My new concerns are that TEM pictures of HTCA in KO spermatids (Fig. 4C) are unclear.I believe Fig. 4C is very critical, but the quality of the enlarged pictures is poor.That's why misunderstandings like the above can happen.I understand the difficulty of observing HTCA structure, but you should work on it until you get suitable pictures.In particular, the HTCA of KO in steps 15-16 is terrible.I think "Dc" becomes an axoneme (centrioles have triplet microtubules without central microtubules) and "Sc" becomes an annulus.I recommend changing the pictures of [13][14][15][16] Related to this concern, Fig. 4E should be modified again.In addition, the cytoplasm of WT sperm in step 13-14, 15-16, sperm and KO sperm in step 13-14, 15-16, sperm is strange.Cytoplasm present below the annulus should be removed (Cytoplasm is present around the flagellum, but not in such large amounts).Also, I'm still uncomfortable because the axonemes are too short.All axonemes or flagellum should extend to the edge of the figures.

#2
The authors mentioned that KO sperm have abnormal mitochondrial sheath arrangements (L499).Fig. 3H shows a crosssection of the midpiece, principal piece and endpiece.But "KO midpiece" appears to be the principal piece.Is this really a midpiece?Furthermore, both endpiece figures still depict the principal piece.You should check the following paper which describes the endpiece features.The endpiece should not have accessory structures.In addition, its legend states that all scale bars are the same size.If so, the midpiece is too small and the endpiece is too large.Given these considerations, the TEM data in this paper cannot be trusted.
I understand the difficulty of finding an endpiece, and I know that many papers incorrectly refer to a midpiece as an endpiece.In my opinion it would be very difficult to count the 9+2 structure of the end piece until a statistical analysis could be done.Based on these results, I also believe Figure 3I has a problem.My suggestion is that it would not make sense to analyze the flagellum separately into three parts, so the abnormal axonemal data in Fig 3I should be combined (midpiece + principal piece + endpiece).In addition, Figure 3H should be changed.When it is difficult to find an endpiece, only the cross-section of the principal piece is sufficient.Since mitochondrial sheaths are so slim in spermatozoa (Fig. 3B and 3E), I also think there might be no mitochondrial sheath in KO spermatozoa.For this reason, mitochondrial staining of spermatozoa is necessary to reveal abnormal mitochondrial sheath arrangements to state L499."Axonemal doublet microtubules can split into two complete singlets in human sperm flagellum tips" by Zabeo et al.
Minor points L145: Manchette is only observed in spermatids."manchette during spermiogenesis and basal plate of spermatozoa" might be better.
L213, 221, Fig. 3B, 3E: A cytoplasmic droplet (CD)-like structure is used by the authors to explain abnormal phenotypes observed in spermatozoa.The cytoplasmic droplet is a normal morphological structure in WT sperm, however."Excess residual cytoplasm" might be suitable for this abnormality.You should check the following paper."An investigation of excess residual cytoplasm in human spermatozoa and its distinction from the cytoplasmic droplet" by Rengan et al.
There is no steps or stages in Fig. 1E and S1.This information is necessary for these figures.Fig. 8D, F, H, J: The authors use "IP:STREP".But I think immunoprecipitation was not performed in these studies.In addition, STREP-IP was used in the figure legend.Fig. 8L: BAG5 does not localize to the basal plate in spermatids with manchettes structure.BAG5 can be localized on the basal plate only during late spermatids and mature spermatozoa (Fig. 1D, 1E, 1F).Therefore, the pink dots (BAG5) on the basal plate in the "Assemble" picture should be removed.Instead, they should be placed on the basal plate in the "Normal spermatozoa" picture.
BAG5 changes their location from manchette to basal plate during spermiogenesis (Fig. 1D, 1E).The authors should mention about that in discussion part.

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if n<5, the individual data points from each experiment should be plotted.Any statistical test employed should be justified.Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name).the assay(s) and method(s) used to carry out the reported observations and measurements.an explicit mention of the biological and chemical entity(ies) that are being measured.an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

Reporting
Adherence to community standards Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Have primary datasets been deposited according to the journal's guidelines (see 'Data Deposition' section) and the respective accession numbers provided in the Data Availability Section?

Data Availability
Were human clinical and genomic datasets deposited in a public accesscontrolled repository in accordance to ethical obligations to the patients and to the applicable consent agreement?

Not Applicable
Are computational models that are central and integral to a study available without restrictions in a machine-readable form?Were the relevant accession numbers or links provided?

Not Applicable
If publicly available data were reused, provide the respective data citations in the reference list.

Not Applicable
The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.Journals have their own policy about requiring specific guidelines and recommendations to complement MDAR.

Fig
Fig. 2G figure includes vas deferens.This information should be included in the figure legend.
Table of Contents for this article are available online." Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section) Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) Provide species, strain, sex, age, genetic modification status.Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID.

In which section is the information available?
Table, Materials and Methods, Figures, Data Availability Section) (Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) If collected and within the bounds of privacy constraints report on age, sex and gender or ethnicity for all study participants.

In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

Checklist for Life Science Articles (updated January Study protocol Information included in the manuscript? In which section is the information available?
ideally, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plotsincludeclearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).a statement of how many times the experiment shown was independently replicated in the laboratory.This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.(ReagentsandTools Table, Materials and Methods, Figures, Data Availability Section)If study protocol has been pre-registered, provide DOI in the manuscript.For clinical trials, provide the trial registration number OR cite DOI.

In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)If sample or data points were omitted from analysis, report if this was due to attrition or intentional exclusion and provide justification.

Sample definition and in-laboratory replication Information included in the manuscript? In which section is the information available?
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)

In which section is the information available?
Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.For publication of patient photos, include a statement confirming that consent to publish was obtained.Not Applicable Studies involving experimental animals: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Include a statement of compliance with ethical regulations.
(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)Studies involving human participants: State details of authority granting ethics approval (IRB or equivalent committee(s), provide reference number for approval.Not ApplicableStudies involving human participants:

Use Research of Concern (DURC) Information included in the manuscript? In which section is the information available?
(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Could your study fall under dual use research restrictions?Please check biosecurity documents and list of select agents and toxins (CDC): https://www.selectagents.gov/sat/list.htmNot Applicable If you used a select agent, is the security level of the lab appropriate and reported in the manuscript?Not Applicable If a study is subject to dual use research of concern regulations, is the name of the authority

granting approval and reference number for
the regulatory approval provided in the manuscript?

and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (